The present application is a continuation in part of US Provisional Patent Application No. 60/013,869 filed Mar. 22, 1996.
1. Field of the Invention
The present invention relates generally to the treatment of cancer. More particularly, it concerns novel compounds useful for chemotherapy, methods of synthesis of these compounds and methods of treatment employing these compounds. The novel compounds are bis-anthracyclines related to anthracyclines such as doxorubicin which is known to have anti-tumor activity.
2. Description of the Related Art
Resistance of tumor cells to the killing effects of chemotherapy is one of the central problems in the management of cancer. It is now apparent that at diagnosis many human tumors already contain cancer cells that are resistant to standard chemotherapeutic agents. Spontaneous mutation toward drug resistance is estimated to occur in one of every 10.sup.6 to 10.sup.7 cancer cells; this mutation rate appears to be independent of any selective pressure from drug therapy, although radiation therapy and chemotherapy may give rise to additional mutations and contribute to tumor progression within cancer cell populations (Goldie et al., 1979; Goldie et al., 1984; Nowell, 1986). The cancer cell burden at diagnosis is therefore of paramount importance because even tumors as small as 1 cm (10.sup.9 cells) could contain as many as 100 to 1,000 drug-resistant cells prior to the start of therapy.
Selective killing of only the tumor cells sensitive to the drugs leads to an overgrowth of tumor cells that are resistant to the chemotherapy. Mechanisms of drug resistance include decreased drug accumulation (particularly in multi-drug resistance), accelerated metabolism of the drug and other alterations of drug metabolism, and an increase in the ability of the cell to repair drug-induced damage (Curt et al., 1984; and Kolate, 1986). The cells that overgrow the tumor population not only are resistant to the agents used but also tend to be resistant to other drugs, many of which have dissimilar mechanisms of action. This phenomenon, called pleiotropic drug resistance or multi-drug resistance (MDR), may account for much of the drug resistance that occurs in previously treated cancer patients.
Gene amplification (i.e., the production of extra copies of genes within a cell) is one of the mechanisms that can lead to drug resistance. Gene amplification is involved in the phenomenon of multi-drug resistance. Multi-drug resistance appears to be linked to over-expression of a cell membrane glycoprotein, termed P-glycoprotein, on the surface of cancer cells (Bell et al., 1985; and Bertino, 1985). The action of this glycoprotein is unknown, but its over-expression is associated with decreased accumulation of multiple chemotherapeutic drugs within the resistant cells. A multi-drug-resistance gene that encodes the P-glycoprotein has been isolated and sequenced, and when it is transferred, this gene confers drug resistance on previously drug-sensitive cells (Gros et al., 1986). The multi-drug-resistance gene termed mdrl is expressed in several normal tissues, and its expression is increased in some human tumors (Fojo et al., 1987). Various human tumors are now being analyzed to determine whether they express this gene. Because many heavily treated patients who are in relapse harbor tumors that do not show over-expression of the mdrl gene, it appears that other mechanisms are probably also involved in causing resistance to chemotherapy.
The commonly used chemotherapeutic agents are classified by their mode of action, origin, or structure, although some drugs do not fit clearly into any single group. The categories include alkylating agents, anti-metabolites, antibiotics, alkaloids, and miscellaneous agents (including hormones); agents in the different categories have different sites of action.
Antibiotics are biologic products of bacteria or fungi. They do not share a single mechanism of action. The anthracyclines daunorubicin and doxorubicin (DOX) are some of the more commonly used chemotherapeutic antibiotics. The anthracyclines achieve their cytotoxic effect by several mechanism, including intercalation between DNA strands, thereby interfering with DNA and RNA synthesis; production of free radicals that react with and damage intracellular proteins and nucleic acids; chelation of divalent cations; and reaction with cell membranes. The wide range of potential sites of action may account for the broad efficacy as well as the toxicity of the anthracyclines (Young et al., 1985).
The anthracycline antibiotics are produced by the fungus Streptomyces peuceitius var. caesius. Although they differ only slightly in chemical structure, daunorubicin has been used primarily in the acute leukemias, whereas doxorubicin displays broader activity against human neoplasms, including a variety of solid tumors. The clinical value of both agents is limited by an unusual cardiomyopathy, the occurrence of which is related to the total dose of the drug; it is often irreversible. In a search for agents with high antitumor activity but reduced cardiac toxicity, anthracycline derivatives and related compounds have been prepared. Several of these have shown promise in the early stages of clinical study, including epirubicin and the synthetic compound mitoxantrone, which is an amino anthracenedione.
The anthracycline antibiotics have tetracycline ring structures with an unusual sugar, daunosamine, attached by glycosidic linkage. Cytotoxic agents of this class all have quinone and hydroquinone moieties on adjacent rings that permit them to function as electron-accepting and donating agents. Although there are marked differences in the clinical use of daunorubicin and doxorubicin, their chemical structures differ only by a single hydroxyl group on C14. The chemical structures of daunorubicin and doxorubicin are shown in FIG. 1.
Unfortunately, concomitant with its anti-tumor activity, DOX can produce adverse systemic effects, including acute myelosuppression, cumulative cardiotoxicity, and gastrointestinal toxicity (Young et al., 1985). At the cellular level, in both cultured mammalian cells and primary tumor cells, DOX can select for multiple mechanisms of drug resistance that decrease its chemotherapeutic efficacy. These mechanisms include P-gp-mediated MDR, characterized by the energy-dependent transport of drugs from the cell (Bradley et al., 1988), and resistance conferred by decreased topoisomerase II activity, resulting in the decreased anthracycline-induced DNA strand scission (Danks et al., 1987; Pommier et al., 1986; Moscow et al., 1988.
Among the potential avenues of circumvention of systemic toxicity and cellular drug resistance of the natural anthracyclines is the development of semi-synthetic anthracycline analogues which demonstrate greater tumor-specific toxicity and less susceptibility to various forms of resistance.
The development of drug resistance is one of the major obstacles in the management of cancer. There are various types of drug resistance, for example, classic MDR as opposed to AD 198 resistance. Furthermore, different cell lines can establish resistance to the same drug in different ways, as seen in the case of the differences in AD 198 resistance in AD 198.sup.R cells as opposed to A300 cells. One of the traditional ways to attempt to circumvent this problem of drug resistance has been combination chemotherapy.
Combination drug therapy is the basis for most chemotherapy employed to treat breast, lung, and ovarian cancers as well as Hodgkin's disease, non-Hodgkin's lymphomas, acute leukemias, and carcinoma of the testes.
Combination chemotherapy uses the differing mechanisms of action and cytotoxic potentials of multiple drugs. Although all chemotherapeutic drugs are most effective on cells that are active in DNA synthesis, many agents--particularly the alkylating agents--can kill cells that are not cycling. Such agents are termed non-cell proliferation-dependent agents can shrink tumor mass by reducing cell numbers; the surviving cells will then move into the cycling compartment, where they are more susceptible to cell proliferation-dependent drugs. The combined use of agents less dependent on the cell cycle followed by those dependent on cell proliferation is effective in enhancing tumor cell death. Each cycle of treatment kills a fixed fraction of cells, so repetitive cycles are required for cure. For example, a drug combination that kills 99.9 percent of cancer cells per treatment cycle would have to be repeated at least six times to eliminate an average tumor burden (if tumor cells did not re-grow between cycles).
Although combination chemotherapy has been successful in many cases, the need still exists for new anti-cancer drugs. These new drugs could be such that they are useful in conjunction with standard combination chemotherapy requires. Or, these new drugs could attack drug resistant tumors by having the ability to kill cells of multiple resistance phenotypes.
A drug that exhibits the ability to overcome multiple drug resistance could be employed as a chemotherapeutic agent either alone or in combination with other drugs. The potential advantages of using such a drug in combination with chemotherapy would be the need to employ fewer toxic compounds in the combination, cost savings, and a synergistic effect leading to a treatment regime involving fewer treatments.
Doxorubicin's broad spectrum of activity against most hematological malignancies as well as carcinomas of the lung, breast, and ovary has made it a leading agent in the treatment of neoplastic disease (Arcamone, 1981; Lown, 1988; Priebe, 1995). Since the discovery of daunorubicin and doxorubicin (FIG. 1), the mechanistic details of the antitumor activity of anthracycline antibiotics have been actively investigated (Priebe, 1995; Priebe, 1995; Booser, 1994).
Although the exact mechanism or mechanisms by which doxorubicin kills cancer cells is still not totally clear, a variety of biochemical evidence implicates nuclear targets in anthracycline-mediated anti-cancer action, those targets being DNA and the DNA-processing enzyme topoisomerase II. Intercalation of anthracyclines into free DNA as well as nucleosomal DNA has been the subjects of numerous reports. In fact, doxorubicin and daunorubicin are now considered to be among the best characterized intercalators, (Chaires, 1990; Chaires, 1990) and the DNA binding affinity of a series of anthracycline antibiotics and their biological activity have been correlated (Valentini et al., 1985). Interference with topoisomerase II activity resulting in protein-linked DNA strand breaks has been linked to anthracycline-induced cell damage. Furthermore, DNA binding has been shown to be necessary for the inhibition of topoisomerase II by anthracyclines (Bodley et al., 1989; Capranico et al., 1990).
Recent studies of anthracycline-DNA interactions have led to a better understanding of the nature of daunorubicin-DNA binding. Investigation of site and sequence specificities revealed that daunorubicin shows preference for binding dGdC-rich regions of DNA that are flanked by A:T base pairs and that the triplet sequences 5'-A/TCG and 5'-A/TGC are preferred binding sites (Chaires et al., 1990; Chaires, 1991; Pullman, 1989; Pullman, 1991; Wang et al., 1990). Interaction of daunorubicin with DNA is probably the best understood of intercalation reactions, thus offering important clues for the rational design of new DNA binding compounds with enhanced binding affinity and sequence selectivity. In this invention the inventors exploit this information to design novel bis-intercalating anthracyclines.
Bifunctional DNA intercalating agents have attracted considerable interest (Wakelin, 1986). Bis-intercalators, both naturally occurring and synthetic, have generally been found to bind more tightly to DNA than mono-intercalators. Bis-intercalators usually have DNA binding constants that are several orders of magnitude larger than those observed for mono-intercalators. The rate of dissociation is generally slower, and consequently, the lifetime of the bis-intercalator-DNA complex is longer than that of a mono-intercalator. Perhaps more importantly, bis-intercalators have larger site sizes than do mono-intercalators. Since the number of DNA base pairs bound to any ligand dictates the possible absolute specificity of the ligand-DNA interaction (Dervan, 1986), a larger site could possibly result in an enhanced sequence selectivity. Still despite the potential of bis-intercalators, the currently known compounds have certain drawbacks
All currently known bisanthracyclines have monomers linked through their C-13 carbonyl functions by a dicarboxylic acid hydrazine bridge (FIG. 2) (Apple et al., 1981; Dawson, 1983; Henry and Tong, 1978; Phillips et al., 1992). However, the carbonyl function at the C-13 position participates in hydrogen bonding interactions that stabilize the drug-DNA complex. Therefore, derivatization at that position should interfere with such interactions so that the monomer units within the bis-intercalator will not retain their optimal binding potential. Thus, previous attempts to synthesize anthracycline bis-intercalators were flawed by a design strategy that used ketone at the C-13 as a linking function, which makes compounds with such a link less active than the parent compound.